Photobiomodulation Therapy (PBMT) Applied in Bone Reconstructive Surgery Using Bovine Bone Grafts: A Systematic Review

The use of low-level laser therapy (LLLT) with biomodulatory effects on biological tissues, currently called photobiomodulation therapy (PBMT), assists in healing and reduces inflammation. The application of biomaterials has emerged in bone reconstructive surgery, especially the use of bovine bone due to its biocompatibility. Due to the many benefits related to the use of PBMT and bovine bones, the aim of this research was to review the literature to verify the relationship between PBMT and the application of bovine bone in bone reconstruction surgeries. We chose the PubMed/MEDLINE, Web of Science, and Scopus databases for the search by matching the keywords: “Bovine bone AND low-level laser therapy”, “Bovine bone AND photobiomodulation therapy”, “Xenograft AND low-level laser therapy”, and “Xenograft AND photobiomodulation therapy”. The initial search of the three databases retrieved 240 articles, 18 of which met all inclusion criteria. In the studies concerning animals (17 in total), there was evidence of PBMT assisting in biomaterial-related conduction, formation of new bone, bone healing, immunomarker expression, increasing collagen fibers, and local inflammation reduction. However, the results disagreed with regard to the resorption of biomaterial particles. The only human study showed that PBMT with bovine bone was effective for periodontal regeneration. It was concluded that PBMT assists the process in bone reconstruction when associated with bovine bone, despite divergences between applied protocols.


Introduction
Low-level laser therapy (LLLT) has been of interest to the scientific community since 1967, when Mester et al. [1] reported its effects on hair growth in rats. It was later verified that this therapy not only stimulated cellular components, but also modulated them, establishing photobiomodulation therapy (PBMT). Further, regenerative medicine has emerged in recent decades to develop adjuvant

Materials and Methods
This systematic review was conducted in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist, as well as previously published systematic reviews [43,44].
For this study, we searched three databases, PubMed/MEDLINE, Web of Science, and Scopus, during September 2019, using the following terms as keywords: "Bovine bone AND low level laser therapy", "Bovine bone AND photobiomodulation therapy", "Xenograft AND low-level laser therapy" and "Xenograft AND photobiomodulation therapy", with no restriction on publication time.
The search results were initially screened by title and then abstract to sort articles into included and excluded folders. Eligibility criteria were applied impartially by the authors regardless of the results presented by each article.
Eligibility criteria: Inclusion criteria were: • Use of bovine bone as a scaffold and PBMT in bone reconstructions; • Human or animal studies; • Publications in the English language only and which allowed full access to the text.

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Each included article should present data regarding: wavelength, output power, energy density, application protocol (points, frequency and days).
Exclusion criteria were: • Duplicate articles; • Excluded because title was not related to aim; • Did not use bovine bone; • Use of other languages (not English); • No access; • Literature review; • Data absence: wavelength (nm), output power (mW); energy density (J/cm 2 ); quantity of radiation.
First, we verified the works that presented titles and abstracts that related to the theme of the initial research, using the two variables: bovine bone as a scaffold and PBMT. The next step was to evaluate and restrict those articles that used bovine bone as a scaffold in animals or humans. The methodology, results and relevance were considered to list the selection of articles.
Analysis and integration of reflective and consistent texts on the subject were performed. The search scheme is presented in Figure 1, according to the PRISMA flow diagram [42,44].

Inclusion of Studies, Quality of Studies, and Test Subjects
The initial search retrieved 240 articles from the three databases, after which 146 articles were excluded because they were duplicated and 36 were excluded due to their titles being unrelated to the theme. The abstracts of 58 articles were read, resulting in the further exclusion of 37 papers as they either did not use bovine bone, did not provide access or were a literature review article, and therefore did not meet the inclusion criteria. This left 21 articles elected for full analysis. After full reading of these 21 articles, three more papers were deleted due to incomplete data. Therefore, in the end, 18 articles related to the theme were included, 17 of which were related to animals and only 1 to humans.
Tables 1 and 2 present the main details of the selected animal and human studies, respectively.

Inclusion of Studies, Quality of Studies, and Test Subjects
The initial search retrieved 240 articles from the three databases, after which 146 articles were excluded because they were duplicated and 36 were excluded due to their titles being unrelated to the theme. The abstracts of 58 articles were read, resulting in the further exclusion of 37 papers as they either did not use bovine bone, did not provide access or were a literature review article, and therefore did not meet the inclusion criteria. This left 21 articles elected for full analysis. After full reading of these 21 articles, three more papers were deleted due to incomplete data. Therefore, in the end, 18 articles related to the theme were included, 17 of which were related to animals and only 1 to humans.
Tables 1 and 2 present the main details of the selected animal and human studies, respectively.  The results of immunohistochemical analysis showed that RANKL expression (>50%, p = 0.199), OPG expression (>75%, p = 0.035) and RANK expression (<50%, p = 0.020) in the experimental group had a significant increase from 7 to 21 days. At 21 days of expression in osteoid formation and bone density in histology (Goldner's trichrome). Histological qualitative analysis (HE and Picrosirius) showed that at 30 days IBB + membrane + PBMT there was more pronounced, well-organized bone formation with dense trabeculae around the graft particles, the cortical repair was complete. All groups irradiated with more collagen fibers. PBMT accelerated bone repair.  Evaluating the 17 articles that involved animal experiments, the total population of test subjects was 663. This total population was made up of 27 rabbits and 636 rats, divided into control groups with a total of 157 animals and intervention groups with 506 animals. The control group animals were always characterized as "empty cavity" or "clot", while the intervention groups contained animals that underwent treatment. Nine studies used male animals [12,15,18,20,22,33,37,46,47] and seven used male and female animals [48][49][50][51][52][53][54], while only one study did not describe the gender of the subjects [45].
The wavelength parameter employed in the studies covered a wide range of values, from 618 to 830 nm. This included one study for each of 618 nm [37], 660 nm [47], 780 nm [22] and 790 nm [18], three studies with 808 nm [15,20,45], two studies with 810 nm [33,36], and nine studies with 830 nm [12,46,[48][49][50][51][52][53][54], as shown in Figure 2. Regarding the type of laser used in the studies, eight studies employed GaAlAs lasers (44.44%), one study cites the use of a light-emitting diode (LED) (5.55%), two specified the use of a diode laser (11.11%) and 7 researches did not mention the type of laser (38.88%). The seven studies that did not Regarding the type of laser used in the studies, eight studies employed GaAlAs lasers (44.44%), one study cites the use of a light-emitting diode (LED) (5.55%), two specified the use of a diode laser (11.11%) and 7 researches did not mention the type of laser (38.88%). The seven studies that did not mention the type of laser used describe the application of the 830nm wavelength, which corresponds to the infrared range ( Figure 3). mention the type of laser used describe the application of the 830nm wavelength, which corresponds to the infrared range ( Figure 3). The energy density employed in the studies ranged from 2 to 354 J/cm 2 , with one study only citing the total energy (24 J/cm 2 ) without specifying the energy per point. Eleven studies used 4 J/cm 2 and two used 6 J/cm 2 , while energy densities of 8.3 J/cm 2 , 30.85 J/cm 2 and 354 J/cm 2 were applied in one study each (Figure 4).  Table 3 presents the outcome measures, characteristics of the test subjects, and results obtained from the studies included in this review. Ten studies evaluated the primary outcome measure of bone The energy density employed in the studies ranged from 2 to 354 J/cm 2 , with one study only citing the total energy (24 J/cm 2 ) without specifying the energy per point. Eleven studies used 4 J/cm 2 and two used 6 J/cm 2 , while energy densities of 8.3 J/cm 2 , 30.85 J/cm 2 and 354 J/cm 2 were applied in one study each (Figure 4). mention the type of laser used describe the application of the 830nm wavelength, which corresponds to the infrared range ( Figure 3). The energy density employed in the studies ranged from 2 to 354 J/cm 2 , with one study only citing the total energy (24 J/cm 2 ) without specifying the energy per point. Eleven studies used 4 J/cm 2 and two used 6 J/cm 2 , while energy densities of 8.3 J/cm 2 , 30.85 J/cm 2 and 354 J/cm 2 were applied in one study each (Figure 4).  Table 3 presents the outcome measures, characteristics of the test subjects, and results obtained from the studies included in this review. Ten studies evaluated the primary outcome measure of bone  Table 3 presents the outcome measures, characteristics of the test subjects, and results obtained from the studies included in this review. Ten studies evaluated the primary outcome measure of bone density using four major methods: µCT, histological analysis of percent volume density of bone (v/v), plain X-rays and the multimodal CMS/SS OCT system. Five studies evaluated the secondary outcome measure of expression of markers, most commonly examining expression of receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG) and receptor activator of nuclear factor-κB (RANK), through histopathological analysis, inflammatory process detection and Raman spectroscopy, and measurement of hydroxyapatite deposition. Table 3. Data from included studies regarding outcome measures, subject attributes, and results.

Authors
Quantitative Analyzis Measurements Results

Discussion
In recent decades, there has been a significant increase in the incidence of craniomaxillofacial and orthopedic disorders, although this has been simultaneous with remarkable progress in the development of biomaterials for reconstruction of lost bone tissue [55].
However, even though there is a wide variety of bone substitutes with satisfactory bone-filling results, histological evidence and biological behavior have only been reported for bovine bone derivatives. Thus, these xenografts have transformed reconstructive surgery and significantly improved clinical outcomes [56].
In addition, noninvasive, adjuvant methods in tissue regeneration have been associated with grafting techniques in an attempt to overcome some practical limits and further improve the repair results of defects filled with biomaterial. Given this context, we performed a review of the scientific literature in order to elucidate the relationship of PBMT with bovine bone when the latter is used as scaffolding for bone reconstruction.
Scientific research related to tissue engineering aims to investigate the process of bone reconstruction using scaffolds, as these are necessary as an auxiliary means for growth of new bone tissue [57]. Efforts to minimize complications and the time needed to heal by improving the process and enhancing biocompatibility has led to the emergence of PBMT-associated biomaterial application in the world literature [12,31]. Bovine bone is listed as the most frequently used type of graft in the literature for the reconstructive bone process [15,20,[36][37][38][39].
Rats accounted for 95.92% of the total animals used in the articles evaluated, showing a preference for these animals in empirical study. One advantage of using rats is their easy handling due to their size, and they are generally chosen for preclinical studies in bone reconstruction biomaterial tests-being the main choice in in vivo studies in regenerative processes [58,59].
The use of male animals in nine of the studies examined suggests a preference of gender for test subjects. This decision is supported in the literature, as it avoids the possible influence of female inhibitory hormones in relation to bone tissue, in addition to the lower risk of fracture and greater bone mass [60,61].
Concerning the use of bovine bone, a preference for its inorganic phase (10 papers) was identified [12,15,20,22,33,37,47,[52][53][54], although no differences in the process of bone healing when associated with a laser were reported, while six studies [18,46,[48][49][50][51] used bone with an organic matrix. Bovine bone matrix has been widely used as a heterogeneous graft in orthopedic surgeries and craniofacial reconstructive procedures with satisfactory osteoconductive properties [32][33][34]. However, previous studies have shown differences between the effectiveness of inorganic and organic bovine matrices in the bone repair process. Some researches advocate for the use of inorganic material due to the absence of proteins and cells, which decreases the risk of immunogenic reactions. Further, this material provides a large amount of hydroxyapatite, which is a major component in normal bones [62]. Other researches elect to use organic material for the permanence of its protein scaffold, mainly comprised of type I collagen, which may initially favor formation of the extracellular matrix [15,63].
During this review, an array of different protocol elements was observed. A range of wavelength parameters-from 618 to 830 nm [12,37,46,[48][49][50][51][52][53][54]-was used, along with variation in energy density, application time and type of laser used, even with similar types of lesions. Most articles used the infrared light spectrum [12,15,18,20,22,33,45,46,48,49,[51][52][53][54], including the study on humans [36], with promotion of new (local) formations and increased protein and genes of osteoblastic factors. PBMT involves radiation from the red to infrared regions, with the latter being most cited in the literature as effective in the early stages of bone repair during the reconstruction process. This is because, at the early stages, there is a large amount of differentiating cells, and reduction of these cells at a late time of repair reduces the PBMT-related osteostimulatory potential [25,48,64].
Regarding the evaluation time of the experiments performed in the analyzed articles, a preference for periods up to 30 days was observed, as the literature shows more modulatory effects of PBMT during the early stages of the bone repair process. Specifically, effects such as greater proliferation of osteoblasts, collagen fibers, and mesenchymal cells, less inflammation, and greater expression of immunomarkers have been reported [12,15,18,20,37,65].
The therapeutic effects of PBMT is dependent on the mode of application, time, frequency and number of sessions of irradiation and dosing, as well as the biologically-dependent relationship of energy density and intensity. PBMT presents conflicting results in the literature, especially with regard to these modulatory effects, as the parameters (wavelength, power density, treatment dose, method and number of applications) are greatly diversified [66][67][68]. When verifying that PBMT has a major effect on mitochondria, the parameter of wavelength appears to have a major influence on the therapeutic process, with the visible (red) wavelengths activating the mitochondrial respiratory chain and the non-visible (infrared) wavelengths acting on the cell membrane. Two experiments with beneficial cellular effects of laser application can be exemplified, where greater collagen production from fibroblasts and osteoid matrix originating from osteoblasts was observed [50,66,69].
The presence of more organized collagen fibers when bovine bone grafts are associated with PBMT has been reported, relating to a biostimulatory effect on collagen production [47][48][49][50][51][52][53][54], as well as improving osteoblastic activity with the release of calcium hydroxyapatite [18,47]. This relationship with osteoblast activity seems to be related to an increase in alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (Runx2) and Jagged1 differentiation genes, and osteocalcin (OCN) [15], up to a period of 30 days. Kim et al. [20] pointed to an increase of receptor activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG) and receptor activator of nuclear factor-κB (RANK) in the first 7 days, as already mentioned in previous studies relating bovine bones and lasers [33,70].
When using PBMT with 660 nm [47] and 618 nm [37], studies mentioned that, despite the increase of new bone, there was no resorption of bovine bone particles, while at 780 nm [22] and 808 nm [15], the biomaterial resorption occurred partially. It has been reported that osteoconductive biomaterials reduce local bone formation, which, by not being absorbed eventually, replace the new bone [63]. Oliveira et al. [15] found 60% more bone in a group without a biomaterial (control); however, computed microtomography showed that, in the groups with bovine bone, there was a greater amount of mineralized tissue. This suggests that, clinically, the use of osteoconductive biomaterials is important for maintaining morphology and function, rather than for new bone formation itself.
Most studies used infrared spectrum wavelengths, with GaAlAs being cited in eight studies [12,15,20,22,36,[45][46][47]. Seven studies [48][49][50][51][52][53][54] did not state which type of laser they used, but did describe the application of 830 nm in the infrared range. The infrared spectrum is the most widely used in reconstructive processes, as it shows less energy loss when penetrating tissues, with about 37% reaching 2 mm deep and, at larger thicknesses, the maximum loss can be as little as 162.92 mW per cm 2 [12,71].
Bovine biomaterial is widely used and has good results in bone reconstruction processes, such as enlargement of the maxillary sinus or preparation for dental implants [72,73]. Of the articles included in this review, 17 cite positive results regarding the association of bovine bone with PBMT. However, Bosco et al. [47] concluded that PBMT stimulates bone formation regardless of the presence of biomaterial. The presence or absence of a membrane plus a biomaterial also did not seem to have an influence on the biostimulatory effects of the laser in three other studies [51,52,54]. This is in contrast to the results reported by Ghahroudi et al. [33], wherein greater bone neoformation was found when it was associated with both a biomaterial and PBMT, and the bovine bone group alone was better than a laser alone.
A lack of persistence in the standardization of methodology employed by authors was observed, with instances of absence of important data, such as output power, energy density and application time, a pattern also observed in reviews relating PBMT to other types of lesions (such as nervous) [5]. It is extremely important to highlight the scarcity of publications addressing PBMT. This complementary treatment method is cited in the literature in association with the widely-used bovine bone scaffolds in bone reconstruction, with both having good results and clinical applicability.

Conclusions
At the end of this review, it can be verified that the data presented in recent literature shows potential to improve the bone reconstructive process using PBMT together with bovine bone as a scaffold. A variability of parameters seems to be common in studies using PBMT, as well as a lack of parameters, generating doubts regarding reproducibility and, consequently, the production of satisfactory results.